Phage displaying system expressing single chain antibody

ABSTRACT

Disclosed are nucleic acid libraries for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and for facilitating production of a disulfide-stabilized single chain antibody. Also disclosed are host cell libraries and phage libraries including the nucleic acid libraries. Further disclosed are methods for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and methods for producing a disulfide-stabilized single chain antibody and non-fusion form thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/897,850, filed on May 20, 2013, which is a divisional of U.S. application Ser. No. 12/854,632, filed on Aug. 11, 2010, which claims priority to U.S. Provisional Application No. 61/232,819, filed on Aug. 11, 2009. The contents of all prior applications are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is related to a phage displaying system expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv).

BACKGROUND OF THE INVENTION

A single chain variable fragment (scFv) is a single polypeptide chain antibody fragment having a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains. An scFv displayed as a fusion protein N-terminal to the pIII minor capsid protein on filamentous phage surface is one of the most prominent methods in antibody engineering. It was reported that the small size of the scFv enabled superior tissue-penetrating capabilities over whole IgG or Fab fragment, making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (Michnick, S. W., and Sidhu, S. S. (2008) Nat Chem Biol 4(6), 326-329).

Yet, under practical application conditions, an scFv scaffold tends to form aggregation. The aggregation has much to do with the stability of the two variable domains and the dimeric interface. The instability of the scFv structure also compromises the fidelity in reproducing the antibody gene products on phage surface, causing biases in favor of more stable scFv molecules over the less stable ones, or selecting non-folded structures on phage surfaces but nevertheless binding to an antigen. This structural instability thus impacts negatively on the applications of scFv in biotechnology and medical uses.

One way to stabilize the scFv scaffold is to engineer a disulfide bond between the two Fv domains, so that the variable domains can be covalently linked with a disulfide bond. Single chain disulfide-stabilized Fv fragment (sc-dsFv) format was constructed in a single polypeptide chain, as in scFv, with a disulfide framework region (Young, N. M. et al., (1995) FEBS Lett 377(2), 135-139; Worn, A., and Pluckthun, A. (1999) Biochemistry 38(27), 8739-8750). The sc-dsFv molecules could be expressed in E. coli, but not be expressed on phage surface or as soluble form secreted by E. coli in a culture medium, mostly due to severely decreased yield because of the introduction of interface cysteines (Worn, A., and Pluckthun, A. (2001) J Mol Biol 305(5), 989-1010).

Up to now, phage-displayed sc-dsFv libraries and their applications have not been established.

BRIEF SUMMARY OF THE INVENTION

The invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression, for which the expression with conventional signal sequences was not viable. The optimized signal sequences and related discovering methodologies led to the establishment of phage display systems with the sc-dsFv format, enabling the demonstration and comparison of the performance of the sc-dsFv phage display platform with that of the conventional scFv platform.

Accordingly, in one aspect, the present invention provides a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of expression constructs, each of which includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b)  (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c)  (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH. Each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

In another aspect, the invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of host cells each containing an expression construct that includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond; the second nucleotide sequence is located 3′ downstream to the first nucleotide; the signal peptide has the amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b)  (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c)  (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH. each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

In another aspect, the invention provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library has a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of the phage. The phage library is prepared by the steps of: providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles; the expression construct that includes a first nucleotide sequence encoding a signal peptide, a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, and a third nucleotide sequence encoding a phage envelop protein; the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence; the signal peptide has the amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b)  (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c)  (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH, each of X₁-X₁₀ in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

In addition, the invention provides a sc-dsFv phage display platform. According to the invention, a large scale screening for optimal signal sequences was carried out. In one example of the invention, the signal sequences that were effective for phage-displayed sc-dsFv and non-fusion soluble sc-dsFv secretion in E. coli Amber suppressor strain ER2738 were screened to obtain the sequence preference patterns emerged from the optimum signal sequences.

In still another aspect, the present invention provides an isolated nucleic acid, having a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The signal peptide has the amino acid sequence of

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

In a further aspect, the present invention provides a host cell containing the nucleic acid described above.

In a further more aspect, the present invention provides a phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface. The phage is prepared by a method having the steps of: providing the above-described host cell, and culturing the host cell in a medium under conditions allowing expression of the phage.

In further another aspect, the present invention provides a method for producing a disulfide-stabilized single chain antibody. The method includes the steps of providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody. The expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond; the signal peptide has the amino acid sequence of:

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

In addition, the present invention provides a new signal peptide that facilitates production of disulfide-stabilized single chain antibody, and the nucleic acid encoding the signal peptide.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 is a schema showing the signal sequence in pCANTAB5E and the constructs of DNA libraries to diversify the tentative signal sequence responsible for the expression of the phage-displayed pIII fusion proteins.

FIG. 2 is a set of diagrams showing the results of the increased binding to VEGF for phage-displayed sc-dsFv signal sequence variants enriched from the three libraries after selection/amplification cycles, including (A) after each round of selection/amplification cycle, the values of the binding of the rescued phage to immobilized VEGF as measured with ELISA. The ELISA signal strengths are shown in the y-axis, as functions of the selection/amplification cycle; and (B) the numbers of output phage particles titered after each round of selection/amplification cycle for each of the three libraries; the output phage titers, as shown in the y-axis, were plotted against the number of the selection/amplification cycles.

FIG. 3 is a schema showing the DNA construct of the S5 anti-VEGF sc-dsFv as a pIII fusion protein in the pCANTAB5E phagemid.

FIG. 4 is a diagram showing VEGF-binding strengths of the phage-displayed anti-VEGF sc-dsFv's from various signal sequence variants with or without fXa digestion. Eight variants with maximal fXa digestion resistance from a 96-well ELISA plate containing 96 randomly picked variants were selected from each of the VEGF-binding enriched libraries after the 4^(th) round of selection/amplification cycle. These variants were cultured and the rescued phages were allowed to bind to immobilized VEGF with (grey histogram) and without (black histogram) the fXa treatment, and the VEGF-binding strengths (y-axis) were measured with ELISA. The error bars were derived from three repeats of the ELISA measurement.

FIG. 5 is a diagram showing the binding strengths of phage-displayed anti-HAs scFv/sc-dsFv. One of the scFv phages with specific binding ability to H5, 8a, and the other one with broad-spectrum ability to HAs, 12a, were engineered to disulfide-stabilized scFv (ds-scFv) formats; the sc-dsFv construct was different from the scFv construct in the mutations (L:Gly100Cys & H:Gly44Cys). Av1 was negative control of an scFv displayed on the phage; and TAA means the phage does not contain any displayed protein; and various HA subtypes were precoated to ELISA wells to determined binding activity, and the error bars were derived from three repeats of the ELISA measurements.

FIG. 6 is a set of diagrams showing correlations between sc-dsFv folding quality and resistance to fXa digestion. (A) shows a comparison of the extents (percentages) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4; both of the axes show the ratio (percent) of the ELISA signal for VEGF-binding after the fXa treatment over the ELISA signal for VEGF-binding before the fXa treatment; the y-axis shows the data from secreted sc-dsFv; the x-axis shows the data from phage-displayed sc-dsFv. (B) shows a comparison of the extents (percentages) of the interface disulfide bond formation (y-axis) with the folding quality (x-axis) of the sc-dsFv from the optimum signal sequence variants from L4. The sc-dsFv folding quality (x-axis) is represented as the sc-dsFv-VEGF binding ELISA signal divided by western blot signal probed with anti-E tag antibody (E/W, VEGF binding signal divided by secreted sc-dsFv quantity), and then the ratio is normalized with that of anti VEGF scFv (fXa+) (CE/CW, VEGF binding signal divided by secreted scFv quantity), that is, the folding quality is quantified with the ratio: (E/W)/(CE/CW); the error bars in each data point indicate the standard deviations from three repeats of the experiment; the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient was shown in each panel.

FIG. 7 is a set of diagrams showing stability test of soluble sc-dsFv; including (A) showing the results of the soluble sc-dsFv incubated at 37° C. as the indicated time shown in the x-axis, and the binding capacities estimated with ELISA against VEGF, shown in y-axis; the ELISA signal was normalized against that of the secreted protein kept at 4° C.; and (B) showing the fXa resistance percentages of the soluble sc-dsFv plotted against the end binding capacities after 12 days of incubation in 37° C.; the error bars in each data point indicate the standard deviations from three repeats of the experiment, and the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient are shown in the panel.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

Amino acids can be expressed by three letters or one letters. Table 1 lists standard amino acid abbreviations.

TABLE 1 Standard amino acid abbreviations Amino Acid 3-Letter 1-Letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Very little is known as to why some sc-dsFv constructs could not be expressed on phage surface, and why the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm. The mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasmic membrane could be a key determinant for the folding. It was unexpectedly found in the invention that for the expression of the displayed protein on the phage surface, alternative sequences in the signal peptide region can modulate the expression level and folding quality of the displayed protein. Accordingly, the invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression. Based on the optimized signal sequences and the methodologies of the invention, phage display systems with the sc-dsFv format are established.

According to the present invention, a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody is provided. The library has a plurality of expression constructs, each of which includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b)  (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c)  (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH, each of X₁-X₁₀ in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

In one embodiment, each of the expression constructs further includes a third nucleotide encoding a phage coat protein, and the third nucleotide sequence being located 3′ downstream to the second nucleotide.

The term “signal peptide” or “signal sequence” used herein refers to a short (i.e. 3-60 amino acids long) peptide chain that directs the transport of a protein. The signal peptide is known to be responsible for the sec system-dependent translocation of the sc-dsFv-pIII fusion from the translation site in cytoplasm across the periplasmic membrane, a critical process for the integration of the displayed protein on the recombinant phage. Considering the vast signal peptide sequence space needed to be explored, the present invention provides biological combinatorial strategies to diversify the signal peptide sequences with synthetic phage display libraries. The variants in the phage libraries were selected and screened for high expression capabilities, so as to identify the key regions of the signal peptide sequences, including the optimal amino acid sequences, positions and types that are effectively responsible for the sc-dsFv expression on phage surface.

The term “single chain variable fragment” or “scFv” used herein refers to a single polypeptide chain antibody fragment construct encoding a first variable region and a second variable region, with a flexible linkage peptide connecting the two domains. The first and the second variable region can be either a light chain or a heavy chain variable region. The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. One shortcoming of the scFv scaffold is the aggregation tendency of the scFv molecules under physiological and storage conditions. The aggregation mechanism has much to do with the stability of the two variable domains and the dimeric interface. This structural instability has thus impacted negatively on the utilities of scFv, leading to uncertainties to the outcomes of the selected and screened scFv molecules in terms of their potential applications in biomedicine.

The term “disulfide-stabilized single chain antibody variable fragment” or “sc-dsFv” used herein refers to a single polypeptide chain containing two variable regions capable of forming an interface disulfide bond, where each of the two variable regions may be a heavy chain variable region or a light chain variable region. According to the invention, the sc-dsFv-pIII fusion protein can be prepared by using the optimal signal sequences capable of directing the sc-dsFv expression on phage surface.

In an embodiment of the invention, the overlapping segments encompassing the complete signal sequence region governing the protein trafficking of the model anti-VEGF sc-dsFv fusion protein were searched with biological combinatorial methodology for sequence preferences leading to effective expression of the sc-dsFv. The engineering platform established for the disulfide-stabilized antibody variable domain fragment as demonstrated could be used to prepare many of scFv molecules in a more stable structure, which could be carried out under harsh conditions, and have longer shelf-life.

According to one embodiment of the invention, to select signal sequences for effective expression of anti-VEGF sc-dsFv on M13 phage surface, phage display libraries L2, L3 and L4 were constructed to diversify the signal sequence as shown in FIG. 1, where M13pIII-pelB indicated the signal sequence being the wild type signal sequence for pIII in M13 phage genome in connection with pelB peptidase cleavage site. The complexities of the L2, L3 and L4 phage display library were 3.1×10⁹, 3.7×10⁹, and 1.5×10⁹, respectively. These libraries were designed to efficiently diversify the signal peptide sequences on identifying the optimum signal peptides for expression sc-dsFv.

In one example of the invention, the expression construct is a phagemid. Among the expression constructs, the nucleotide sequence of the signal peptide, sc-dsFv and the phage coat protein could be operatively linked in a random order. In one preferred example of the invention, the second nucleotide sequence encoding sc-dsFv is located 3′ downstream to the first nucleotide encoding the signal peptide, and the third nucleotide sequence encoding the phage coat protein is located 3′ downstream to the second nucleotide sequence.

In one embodiment of the invention, a sc-dsFv library, containing more than one billion sc-dsFv variants, is propagated with an E. coli vector of bacterial phage origin following the method as described by McCafferty, J. et al. (Nature 348(6301), 552-554, 1990). The recombinant phages displaying the sc-dsFv variants can be selected or screened for antigen-binding and re-amplified with the host cells, i.e. E. coli.

Furthermore, the present invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of host cells each containing the aforementioned expression constructs.

The present invention also provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of said phage. The phage library is prepared by the steps of: providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles. The expression construct includes (1) a first nucleotide sequence encoding a signal peptide, (2) a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, and (3) a third nucleotide sequence encoding a phage envelop protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence. The signal peptide has the amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b)  (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c)  (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH, each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

On the other hand, a sc-dsFv engineering platform is established for preparation of scFv molecules in a more stable structure in the present invention. Accordingly, the present invention provides an isolated nucleic acid that has a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁ is A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

According to the invention, the nucleic acid further includes a third nucleotide encoding a phage coat protein. The third nucleotide sequence is located 3′ downstream to the second nucleotide sequence.

In one example of the invention, anti-VEGF sc-dsFv phage display platform was developed. As shown in FIG. 1, expression constructs for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody were designed. Each of the three DNA libraries (L2, L3, and L4) contained ten consecutive NNK degenerate codons covering overlapping regions around the signal sequence. N stands for A, G, T, or C, 25% each; K stands for G or T, 50% each. The NNK degenerated codon represents 32 possible triplet combinations, encoding all 20 natural amino acids and an amber stop codon (TAG). Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (see FIG. 1) can all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.

In order to further identify binding variants, more than 3000 colonies were randomly selected from each of the libraries L2, L3, and L4 after selection/amplification cycles for enrichment of the binding variants. These phage colonies were individually rescued and spotted on nitrocellulose membranes coated with VEGF (100 μg/30 ml). According to the invention, each of the signal peptides having the amino acid sequences of SEQ ID NOS: 5-593 as listed in Tables 2, 3 and 4 was obtained and proved to be capable of facilitating the expression of the sc-dsFv on phage surface. After normalization based on the standard phage solution signals in each of the blocks, the phage-displayed scFv expression efficiency for each of the samples was calculated with the following equation:

${Ratio} = {\frac{{sample}({CV})}{{sample}\left( {C\; 0} \right)}/\frac{{control}({CV})}{{control}\left( {C\; 0} \right)}}$

The value of the sample (CV) is the average normalized signal from VEGF-coated membrane; that of the sample (C0) is the averaged normalized signal from the un-coated and un-blocked membrane. Similarly, those of the control (CV) and control (C0) are the averaged normalized signals for the control phage in the same block where the sample signals are measured on corresponding membrane. The ratio derived from the equation was used to rank the efficiency of the sample phage binding to the immobilized VEGF. All the phage samples with measurable binding strengths with the immobilized VEGF were ranked; the signal sequences of the top fifty ranked phage samples are shown and marked with “*” in Tables 2-4.

Accordingly, new signal peptides that facilitate production of disulfide-stabilized single chain antibody were obtained (see Example 2). In the embodiment of the invention, the signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-593 were proved to facilitate production of disulfide-stabilized single chain antibody. On the other hand, a new isolated nucleic acid encoding the above mentioned signal peptide was provided as well.

In a preferred embodiment of the invention, the signal peptide selected from the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 381-429 and 476 was obtained and proved to facilitate production of disulfide-stabilized single chain antibody. Accordingly, the preferred isolated nucleic acid encoding each signal peptide as mentioned was also provided.

In one example of the present invention, the anti-VEGF sc-dsFv was developed by using the signal peptides as identified and obtained by the method of the present invention. In another example, anti-H5 sc-dsFv against influenza virus was developed (see FIG. 5).

In order to confirm the formation of disulfide bond in the phage-displayed sc-dsFv variants of the present invention, a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains was constructed. As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa−) bound to immobilized VEGF. In contrast, with the fXa treatment, only the anti-VEGF scFv(fXa−) bound to immobilized VEGF. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. The anti-VEGF scFv(fXa−) was quite insensitive to the treatment of fXa, indicating that no other fXa substrate sequences exist in the displayed protein.

Unexpectedly, it was found in the present invention that each of the signal peptides having the amino acid sequences of SEQ ID NOS: 5-593 as listed in Tables 2-4 enabled the expression and proper folding of the sc-dsFv structure on the phage-displayed platform. In addition, they resulted in secretion of the soluble non-fusion sc-dsFv in culture media.

Accordingly, the present invention also provides a method for producing a disulfide-stabilized single chain antibody. The method includes providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody. The expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3′ downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W; X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in which X₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

Similar to the aforementioned experiment, the extent (percentage) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4 were tested. As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule. As shown in FIG. 6B, the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction.

In the present invention, a stability test of soluble sc-dsFv was conducted. As shown in FIGS. 7A and 7B, the sc-dsFv antibody fragment scaffold was indeed substantially more stable than the scFv scaffold due to the interface disulfide bond in the sc-dsFv constructs.

According to the invention, the concentration of sc-dsFv antibody produced by the method disclosed herein was unexpectedly high, and stable. Thus, the present invention provides the sc-dsFv at a high concentration sufficient for coating on a solid phase to produce an array for detection or diagnosis without aggregation, different from the prior art where sc-Fv tends to precipitate under the same concentration due to aggregation.

Accordingly, the present invention provides an array of disulfide-stabilized single chain antibodies produced by the aforementioned method coated on a solid phase. In one example of the invention, the solid phase may be made from silicon, plastic, nylon, glass, ceramic, photoresist or rubber. In one embodiment of the present invention, a microarray test was established using the disulfide-stabilized single chain antibodies produced by the method of the invention, demonstrating that influenza virus could successfully be detected by an array of a serious dilution of anti-H5 sc-dsFv coated on a glass.

The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

EXAMPLES Preparation 1: VEGF Expression and Purification—Human VEGF-121

Human VEGF-121 (VEGF-A residue 34-135 receptor binding domain) (Fuh, G. et al., (2006). J. Biol. Chem., 281, 6625-6631) was expressed in E. coli as inclusion body. The refolding and purification of VEGF-A were carried out as described in Chang, H. J., et al., (2009) Structure, 17, 620-631.

Preparation 2: Phage Display Libraries with Diversified Signal Sequences N-Terminal to the sc-dsFv-pIII Fusion Protein

Phage display libraries with diversified sequences in the signal peptide region N-terminal to the sc-dsFv-pIII fusion protein were constructed with pCANTAB5E phagemid (GE-Amersham Biosciences) as shown in FIG. 1. Primers encoded with the sequence diversification shown in FIG. 1 were synthesized by IDT (Integrated DNA Technologies).

For each of the phage display libraries, phagemid templates were constructed with TAA stop codons inserted in the sequence region for diversification (Huang et al., (2010) J. Biol. Chem., in press). The M13pIII-pelB signal sequence for phage-displayed pIII-fusion protein is a combination of the wild-type M13 signal peptide N-terminal to gene III (MKKLLFAIPLVVPFYSHS) (SEQ ID NO:594) and the pelB signal sequence of Pectobacterium wasabiae (MKYLLPTAAAGLLLLAAQPAMA) (SEQ ID NO:595). This merged signal sequence (shown in bold font above) was considered containing the tentative n- h- and c-regions of the signal sequence. DNA libraries were constructed to diversify the amino acid sequence in the key regions. Each of the four of DNA libraries (L2, L3, L7) contained ten consecutive NNK (N stands for 25% of G, C, A, and T, and K stands for 50% of G and T; underlined by dashed lines) degenerate codons covering a portion of the tentative signal sequence. Also shown in the Figure are the sequences containing TAA stop codons (underlined regions) used as the templates for the library constructions. The oligonucleotide-directed mutagenesis procedure initially proposed by Kunkel (Kunkel et al., (1987) Methods Enzymol, 154, 367-382) was used for the phagemid library construction. The TAA stop codons in the phagemid templates ensure that the un-mutated phagemid templates after the mutagenesis procedure are incapable of producing pIII fusion protein for phage surface display (Sidhu and Weiss, (2004) Construction phage display libraries by oligonucleotide-directed mutagenesis. In: Clackson, T., and Lowman, H. B. (eds). Phage Display, 1st Ed., Oxford University Press, New York).

After the oligonucleotide-directed mutagenesis procedure, E. coli strand ER2738 was transformed with the phagemid libraries and the recombinant phage particles were rescued with helper phage M13KO7 (GE-Amersham Biosciences). The phage particles were precipitated with PEG/NaCl, and resuspended in PBS. More details of the phage library preparation can be found in a previous publication (Hsu, H. J. et al., (2008) J Biol Chem 283(18), 12343-12353).

Seven sc-dsFv variants were constructed on the basis of the phagemid encoding the template anti-VEGF scFv(fXa+): S1(L:Gln38Cys & H:Gln39Cys); S2(L:Gly41Cys & H:Gly42Cys); S3(L:Ala43Cys & H:Gln112Cys); S4(L:Phe98Cys & H:Leu45Cys); S5(L:Gln100Cys & H:Gly44Cys); S6(L:Gln38Cys & H:Leu45Cys); S7(L:Ala43Cys & H:Gln112Cys & L:Gln100Cys & H:Gly44Cys). These cysteine pairs were determined by distance constrain for possible disulfide bonds in the model structure (PDB code: 2FJG).

Preparation 3: Biopanning Against VEGF with Phage-Displayed Anti-VEGF sc-dsFv Libraries

Maxisorb Immune Tubes (Nunc) were coated with VEGF (25 μg in 1 ml PBS in each tube) at 4° C. overnight. The tubes were blocked with 4 ml of 5% skim milk in PBST (PBS with 0.05% Tween 20) for one hour at room temperature with gentle shaking and then washed with PBST. In each of the tubes, 10¹¹ colony-forming units (cfu) of phage from each of the phage display libraries were mixed with 1 ml of 5% skim milk. The phage particles were allowed to bind to the immobilized VEGF in the tube at room temperature for two hours under gentle shaking. After the binding, the tubes were washed 10 times with PBST and 2 times with PBS. One milliliter of E. coli strand ER2738 in the log phase was added to each of the tubes at room temperature with gentle shaking for 15 minutes. From each tube, the infected E. coli was transferred to 10 ml of a 2YT medium containing 20 μg/ml of ampicillin and was titered with 2YT agar plates containing 100 μg/ml of ampicillin. The infected E. coli was incubated at 37° C. for one hour with vigorous shaking Ampicillin was then added to reach final concentration of 100 μg/ml. The culture was incubated for another hour at 37° C. before transferred to final 100 ml 2YT medium (100 μg/ml of ampicillin) containing 10¹¹ cfu M13KO7 helper phage. After two hours of incubation, kanamycin was added to final concentration of 70 μg/ml. The culture was incubated at 37° C. overnight with vigorous shaking. The phage in the supernatant of the culture was harvested by centrifugation. The phage was titered, precipitated with PEG/NaCl, and resuspended in PBS. The phage solution was ready for the next round of selection.

Preparation 4: Enzyme-Linked Immunosorbant Assay (ELISA) for Phage-Displayed Anti-VEGF sc-dsFv Binding Against Immobilized VEGF and Anti-E-Tag Antibody

Single E. coli colonies harboring the selected phagemids were randomly picked using a GENETIX Qpix II colony picker to 96-well deep well culture plates. Each well contained 960 μl 2YT (100 μg/ml of ampicillin and 10 μg/ml of tetracyclin). The culture plates were incubated at 37° C. shaking vigorously for 4 hours before adding 20 μl of M13KO7 helper phage (10¹¹ cfu/ml). The plates were then incubated at 37° C. for one hour with vigorous shaking before adding 20 μl of kanamycin to the final concentration of 50 μg/ml. After overnight incubation at 37° C. with vigorous shaking, the cultures were centrifuged at 3000 g for 10 minutes at 4° C. From each well of the culture plates, 100 μl of the supernatant was mixed with 100 μl of 5% skim milk. Half of the phage mixture was added to a corresponding well of a 96-well Maxisorb microtiter plate precoated with VEGF (1 μg/well) and blocked with 5% skim milk; the other half was added to a corresponding well of another microtiter plate precoated with polyclonal goat anti-E-tag antibody (1 μg/well, Novus Biologicals). After one hour incubation at room temperature, the ELISA plates were washed six times with PBST. The phage particles remained on the plates were measured with HRP-labeled mouse anti-M13 antibody (1/3000, GE Healthcare) and TMB substrate (KPL). The reaction was stopped with 50 μl of 1 N HCl and the signal intensity was measured at OD 450 nm.

Preparation 5: Measurement of Interface Disulfide Bond Formation in Phage-Displayed Anti-VEGF sc-dsFv

Fifty microliters of a freshly prepared phage supernatant (see above) was mixed with 50 μl of a two-fold concentrated reaction buffer containing 1 unit of bovine factor Xa (fXa) (Novagen) in a Maxisorb microtiter plate precoated with VEGF (1 μg/well) and blocked with 5% skim milk. After two hours of enzymatic reaction at 37° C., the phage particles remained bound to the microtiter plate were measured following the same ELISA procedure as described above.

Preparation 6: Western Blot Assay for the Phage-Displayed Anti-VEGF sc-dsFv

Single colony phage was amplified, harvested, precipitated with PEG/NaCl, and resuspended in PBS (see above). Phage particles (10¹¹ cfu) were prepared under either a non-reducing or reducing condition before electrophoresis in a 10% SDS-polyacrylamide gel. After the electrophoresis, the proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with a monoclonal mouse anti-pIII antibody (1/3000 mg/ml, New England Biolabs) for one hour at room temperature. After three washes (5 minutes each) with PBST, the membrane was incubated with HRP-labeled anti-mouse antibody (1/3000, GE Healthcare) for 1 hour at room temperature. After three washes with 10 ml PBST, the membrane was developed with 4-chloro-1-naphthol (4CN) substrate (KPL) until the desired color intensity was achieved.

Preparation 7: Preparation of Non-Fusion Soluble scFv/sc-dsFv

Seven hundred and fifty microliters of mid-log phase (OD_(600 nm)=0.6) E. coli host (non-suppressor strain HB2151 or suppressor strain ER2738) grown in a 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0) was infected with 50 μl of a phage solution (10¹¹ cfu/ml). After one hour incubation at 37° C. with shaking, 100 μl ampicillin in a 2YT medium was added to the final concentration of 100 μg/ml. After another hour of incubation, 100 μl isopropyl-beta-D-thiogalactopyranoside (IPTG) in a 2YT medium was added to the final concentration of 1 mM. The culture was kept at 37° C. with vigorous shaking overnight. The secreted soluble scFv or sc-dsFv in the supernatant was separated from the bacterial host by centrifugation at 3000 g for 10 minutes.

Preparation 8: ELISA for Immobilized VEGF Binding

For phage ELISA, each well in a Maxisorb 96-well microtiter plate (Nunc) was coated with 2 μg VEGF at 4° C. overnight. The wells were blocked with 5% skim milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.1% tween20, pH 7.4) for one hour. After 3×300 μl PBST and 2×300 μl PBS washes, 100 μl of a phage solution and 100 μl of 5% skim milk in PBST were added to each well and incubated at room temperature with shaking for one hour. After washing each of the wells three times with 300 μl of PBST each and twice with 300 μl of PBS each, the bound phages were labeled with anti-M13 antibody conjugated with HRP (GE-Amersham) 1/3000 dilution in 5% skim milk in PBST for one hour. The ELISA signal was developed by incubating each well with 100 μl of a TMB solution (KPL Inc.) for 5 minutes. The reaction was stopped with 100 μl N HCl, and the optical density was recorded with VICTOR3 Multilabel Plate Readers (Perkin Elmer) at 450 nm.

For scFv or sc-dsFv ELISA, 100 μl of a soluble scFv solution was used instead of phage solution, and HRP-conjugated protein L (0.5 μg/ml in 5% skim milk in PBS, from Pierce) was used instead of HRP-conjugated anti-M13 antibody. When needed, the ELISA signals were normalized with the signals of the control anti-VEGF scFv in serial dilution.

Preparation 9: fXa Protease Digestion

For phage solutions, 20 μl (1 unit) of bovine factor Xa (fXa) protease (Novagen) in a six-fold concentrated reaction buffer was added to 100 μl of a phage solution at 37° C. After 2 hours of enzymatic reaction, 100 μl of 5% skim milk in PBST was added to the reaction mixture before the VEGF-binding ELISA measurement was carried out in the manner described in the previous section. The fXa resistance percentage was calculated with the ratio of the ELISA reading in the presence of fXa over the ELISA reading in the absence of fXa. The ELISA readings for the ratio were adjusted by shift the baseline determined with the null control ELISA readings. For soluble scFv/sc-dsFv fXa digestion, all procedures were the same except that the enzymatic reaction was carried out for one hour at room temperature.

Preparation 10: Construction of Anti-H5 sc-dsFv Against Influenza Virus

The construction of scFv library derived from mouse spleen after immunization of hemagglutinin from influenza virus was based on the protocols described in “Phage Display, A Laboratory Manual, edited by Carlos F. Barbas Ill, Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA”. In brief, after hemagglutinin immunization, the total RNA derived from mouse spleen was purified by Trizol reagent (Invitrogen) according to the manufacturer's protocols. After cDNA synthesis by reverse transcriptase, the gene fragments encoded heavy and light chains of antibody variable region were amplified by the specific primer sets described in the book mentioned above, respectively. The scFv fragments were synthesized by two-steps PCR reactions, and then cloned into a pCANTAB 5E phagemid vector with the signal sequence derived from the library 2 for sc-dsFv phage production. The library complexity was 4.5×10⁷. After panning against H5, two clones were selected for mono-spectral binding to H5 (clone 8a) and broad spectral binding to H1, H3 and H5 (clone 12a). These two clones were subjected to disulfide bond formation mutants between L100 and H44 (based on Kabat numbering) and then for sc-dsFv phage production and ELISA detection (8aS5 and 12aS5, respectively).

Preparation 11: Microarray Test for sc-dsFv Binding of H5 Influenza Virus

The just-described sc-dsFv ds-8a or and ds-12a was subcloned into a pET32a vector with thioredoxin as a fusion protein partner at the N-terminus. The thioredoxin-sc-dsFv fusion proteins could be expressed in Rosetta-gami B strain of E. coli in a soluble form. After purification and TEV protease digestion to remove thioredoxin, the purified ds-8a protein was found to have a binding affinity and specificity similar to those of H5 based on an ELISA assay and array studies. The protein ds-AV1, ds-12a and ds-8a were spot on glass slides coated with streptavidin as the purified ds-AV1, ds-12a and ds-8a proteins contained a biotinylated Avitag sequence at their C-termini. The highest protein concentration used for this protein array was 8 mg/ml, 8 mg/ml, and 0.8 mg/ml, respectively. These proteins were 2 fold dilution with a 100 mM sodium phosphate buffer, pH 8.5 from the highest protein concentration for 15 times for spotting (10 nl/spot), and then the protein of each concentration was spotted for 5 replicates. After spotting, each glass slide was sealed to form 16 distinct squares for reaction. After blocking with 5% BSA for 30 minutes, the H5 influenza virus (about ˜10⁷ PFU/ml) was added to react with spotted sc-dsFvs for 30 minutes. After 3 times wash with a phosphate buffer for 5 minutes each, 40 nm fluorescence beads coated with ds-8a (˜10⁷) were added to each square and incubated for 30 minutes. After 3 times wash again with a phosphate buffer for 5 minutes each, the glass array was air-dried for detection.

Example 1 Selection of Signal Sequences for Effective Expression of Anti-VEGF sc-dsFv on M13 Phage Surface

Phage display libraries L2, L3, and L4 were constructed to diversify the signal sequence of the S5 anti-VEGF sc-dsFv-pIII fusion protein as shown in FIG. 1. The complete DNA construct and the amino acid sequence of the S5 anti-VEGF sc-dsFv are shown in FIG. 1. The S5 sequence remained unchanged in all the variants from the libraries. The complexities of the L2, L3, and L4 phage display libraries were 3.1×10⁹, 3.7×10⁹, and 1.5×10⁹, respectively. These libraries were designed to diversify the signal peptide sequences in the h-region, c-region, and a few N-terminal residues of the mature phage-displayed anti-VEGF sc-dsFv.

Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. After four rounds of selection/amplification cycle, the VEGF-binding phage variants were enriched for more than one order of magnitude. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (FIG. 1) could all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.

Example 2 Interface Disulfide Bond Formation in Anti-VEGF sc-dsFv on M13 Phage Surface

In order to test the formation of the disulfide bond in the phage-displayed sc-dsFv variants, we constructed two control phage-displayed anti-VEGF scFv variants: one with a factor Xa cutting site, -IEGR- (SEQ ID NO:599), encoded in the linker peptide connecting the two variable domains (anti-VEGF scFv(fXa+)); the other without this fXa cutting site (anti-VEGF scFv(fXa−)). As shown in FIG. 3, the S5 anti-VEGF sc-dsFv was constructed with a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. Both phage-displayed scFv's did not have the engineered interface disulfide bond as in S5; the scFv(fXa+) construct had the -IEGR- (SEQ ID NO:599) site in the linker peptide (-(G)₄SIEGRS(G)₄S-) (SEQ ID NO:600), while the scFv(fXa−) construct had the conventional -(G)₄S(G)₄S(G)₄S- (SEQ ID NO:601) linker peptide.

As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa−) bound to immobilized VEGF. But with the fXa treatment, only the anti-VEGF scFv(fXa−) bound to immobilized VEGF. In contrast, all the S5 signal sequence variants for the phage-displayed sc-dsFv showed substantial increase in resistance to fXa protease activity, indicating that the interface disulfide bonds in the anti-VEGF sc-dsFv's were formed to stabilize the functional dimeric structure after the cleavage of the peptide linker between the two variable domains. The results unambiguously demonstrated that the engineered interface disulfide bond was correctly formed in the phage-displayed S5 anti-VEGF sc-dsFv from some of the signal sequence variants from all three VEGF-binding enriched signal sequence libraries (L2, L3 and L4).

Example 3 Preference Sequence Patterns of the Optimum Signal Peptides in Effective Expression of Functional Anti-VEGF sc-dsFv

The functionality of the anti-VEGF sc-dsFv on phage surface was quantified with two quantitative measurements: the affinity of the sc-dsFv against VEGF and the extent of the interface disulfide bond formation. After the tests, 250, 126, and 213 optimum signal sequences were found in L2, L3, and L4 library, respectively, which are summarized as following Tables 2-4. Among them, fifty signal sequence variants with the highest sc-dsFv-VEGF binding affinities selected from more than 3000 random single colonies of the enriched libraries L2, L3, and L4 were marked with the symbol “*.” The symbol “q” indicated that the nucleotide sequence TAG (amber stop codon) that could be translated to Gln (Q) with 0.8˜20% in E. coli amber suppressor strains which were normally used in phage production.

TABLE 2 Preference sequence patterns selected from L2 S5 sc-dsFv library No.  Code Sequence SEQ ID NO: M13-pe1B VKKLL FAIPLVVPFY AAQPAMAHHHHHH 4   1 1.12B VKKLL VLSHLPFMTD AAQPAMAHHHHHH * 5   2 9.26.10B VKKLL SHWLLSSqLQ AAQPAMAHHHHHH * 6     3 2.12A VKKLL AMSLAPSVFP AAQPAMAHHHHHH * 7   4 9.12A VKKLL WSLFFqqLNP AAQPAMAHHHHHH * 8   5 2.12F VKKLL LLSLLQRPLP AAQPAMAHHHHHH * 9   6 1.2H VKKLL LSSWLMTRFP AAQPAMAHHHHHH * 10   7 6.9G VKKLL VLSHFPAFVP AAQPAMAHHHHHH * 11   8 1.8F VKKLL PLLSLPLPPN AAQPAMAHHHHHH * 12   9 7.1B VKKLL VLTPMHFSSP AAQPAMAHHHHHH * 13  10 9.26.10A VKKLL ILALPQSYPL AAQPAMAHHHHHH * 14  11 5.4A VKKLL qALYFSLPSS AAQPAMAHHHHHH * 15  12 YJ2.2 VKKLL VSAMTSASFP AAQPAMAHHHHHH * 16  13 5.2F VKKLL LPASWLFGQP AAQPAMAHHHHHH 17  14 10.2D VKKLL WSLFFqqLNP AAQPAMAHHHHHH * 18  15 YJ2.34 VKKLL VVMALRSSAP AAQPAMAHHHHHH * 19  16 3.3F VKKLL FLWPFYNGHI AAQPAMAHHHHHH 20  17 4.1A VKKLL QSFYLSLqLD AAQPAMAHHHHHH * 21  18 10.7H VKKLL SLTFPFTIHS AAQPAMAHHHHHH * 22  19 1.9D VKKLL WPVLSPSLFP AAQPAMAHHHHHH * 23  20 5.12D VKKLL PWLFSTFPSS AAQPAMAHHHHHH * 24  21 1.8D VKKLL IMSSLPTLSP AAQPAMAHHHHHH * 25  22 4.11F VKKLL IMSRVLAPDF AAQPAMAHHHHHH * 26  23 1.7C VKKLL FDFWFSSFLq AAQPAMAHHHHHH * 27  24 4.8G VKKLL YGqLMLLSSD AAQPAMAHHHHHH * 28  25 4.4E VKKLL PWLFPFHAYP AAQPAMAHHHHHH * 29  26 1.12G VKKLL LVMTLSRQPF AAQPAMAHHHHHH 30  27 4.8A VKKLL ASAYLYHGLS AAQPAMAHHHHHH * 31  28 4.4C VKKLL PFFAGVLqHP AAQPAMAHHHHHH * 32  29 3.11A VKKLL ALSSPFFHIP AAQPAMAHHHHHH * 33  30 10.3F VKKLL PTRqPMMYPP AAQPAMAHHHHHH * 34  31 YJ2.15 VKKLL QLLMPFLNSP AAQPAMAHHHHHH * 35  32 9.9H VKKLL CSLGYACIPP AAQPAMAHHHHHH * 36  33 4.9C VKKLL LMPWLFNSPP AAQPAMAHHHHHH 37  34 3.12B VKKLL LDqLAYAALS AAQPAMAHHHHHH * 38  35 4.10G VKKLL qSTVFFSWLS AAQPAMAHHHHHH * 39  36 YJ2.18 VKKLL LPWALSHQVL AAQPAMAHHHHHH * 40  37 7.2E-q VKKLL ALTYPAFLYD AAQPAMAHHHHHH * 41  38 1.11A VKKLL AMAPPMMSMN AAQPAMAHHHHHH * 42  39 5.3D VKKLL WWSSLFAPSP AAQPAMAHHHHHH 43  40 4.6H VKKLL GSFILARSMD AAQPAMAHHHHHH 44  41 5.11C VKKLL MVLTSWHPYP AAQPAMAHHHHHH * 45  42 2.8C VKKLL FSLRFFFPSS AAQPAMAHHHHHH 46  43 2.5F VKKLL WLWSTPLFPH AAQPAMAHHHHHH 47  44 2.2A VKKLL PLLFSLDGDP AAQPAMAHHHHHH * 48  45 3.2C-d VKKLL SVSLSSYSFY AAQPAMAHHHHHH * 49  46 3.1H VKKLL LNGTESAqLF AAQPAMAHHHHHH * 50  47 6.4A VKKLL WHVLPYLPNS AAQPAMAHHHHHH * 51  48 4.10E VKKLL SIVPLFSPqS AAQPAMAHHHHHH * 52  49 7.4H VKKLL VMTSPMLAPG AAQPAMAHHHHHH * 53  50 2.5H VKKLL VLSLPSIAPH AAQPAMAHHHHHH 54  51 6.4E VKKLL qSLLLLRALL AAQPAMAHHHHHH * 55  52 2.1A VKKLL FSLPVFFDLP AAQPAMAHHHHHH 56  53 4.11D VKKLL LLFSMARPLP AAQPAMAHHHHHH * 57  54 7.10A VKKLL TqAVFPFTFN AAQPAMAHHHHHH * 58  55 3.2E VKKLL LASWLFRADM AAQPAMAHHHHHH * 59  56 5.2E VKKLL PFLFPFPSPS AAQPAMAHHHHHH * 60  57 YJ2.128 VKKLL ALSAWSLSQT AAQPAMAHHHHHH * 61  58 4.7H VKKLL ALLPLFPTqH AAQPAMAHHHHHH * 62  59 2.10F VKKLL AALASFPPAP AAQPAMAHHHHHH * 63  60 YJ2.22 VKKLL LLMPFLNQSP AAQPAMAHHHHHH * 64  61 7.5A VKKLL FTSGLKLVPP AAQPAMAHHHHHH 65  62 6.10F VKKLL LqPLLSIYLN AAQPAMAHHHHHH 66  63 4.11B VKKLL LSSLWSAYMD AAQPAMAHHHHHH 67  64 2.5C VKKLL LLGqSLMHFQ AAQPAMAHHHHHH 68  65 YJ2.25 VKKLL PQLAMSLPSI AAQPAMAHHHHHH 69  66 10.3H VKKLL YETMLSSYLY AAQPAMAHHHHHH 70  67 3.10D VKKLL SLYYFPLVPY AAQPAMAHHHHHH 71  68 4.7C VKKLL qRTVAAAYFW AAQPAMAHHHHHH 72  69 4.12D VKKLL FLTWLRYGFP AAQPAMAHHHHHH 73  70 6.1A VKKLL LLLTLMqPTS AAQPAMAHHHHHH 74  71 8.10C VKKLL FDFFTHVHLF AAQPAMAHHHHHH 75  72 5.6E VKKLL ALYPHFVSFT AAQPAMAHHHHHH 76  73 4.11E VKKLL LPYAIqLFSP AAQPAMAHHHHHH 77  74 YJ2.5 VKKLL WFPLHSSLLP AAQPAMAHHHHHH 78  75 4.7A VKKLL PALLLATAAF AAQPAMAHHHHHH 79  76 3.11C VKKLL LASVAWNLDS AAQPAMAHHHHHH 80  77 YJ2.121 VKKLL VGSLLFWPQQ AAQPAMAHHHHHH 81  78 4.5F VKKLL SPLLFLqNYT AAQPAMAHHHHHH 82  79 3.2F VKKLL SYWLDFIqVL AAQPAMAHHHHHH 83  80 10.3C VKKLL VPSFLLSPSP AAQPAMAHHHHHH 84  81 9.23.7H VKKLL SLYWLTSqPL AAQPAMAHHHHHH 85  82 3.9A VKKLL FALSSVHSPP AAQPAMAHHHHHH 86  83 4.11H VKKLL SYYSLLYSYP AAQPAMAHHHHHH 87  84 3.1C VKKLL LVSGLqPWYF AAQPAMAHHHHHH 88  85 2.5A VKKLL VLATPLHLSP AAQPAMAHHHHHH 89  86 10.6H-q VKKLL SLAFPLFTPP AAQPAMAHHHHHH 90  87 3.6A VKKLL SLVPIFPFST AAQPAMAHHHHHH 91  88 8.10D VKKLL qPVLFSFFIR AAQPAMAHHHHHH 92  89 4.3B VKKLL MSqFLNLLSP AAQPAMAHHHHHH 93  90 2.3G VKKLL WAVqPLFPLN AAQPAMAHHHHHH 94  91 5.3H VKKLL MFSLVPSPPI AAQPAMAHHHHHH 95  92 10.7B VKKLL PFFLQPFqFP AAQPAMAHHHHHH 96  93 7.2D-q VKKLL PDLLASVLPV AAQPAMAHHHHHH 97  94 2.9H VKKLL FWqFLWPSLP AAQPAMAHHHHHH 98  95 6.4A VKKLL LLGqFFPNPM AAQPAMAHHHHHH 99  96 6.4D VKKLL TLSALSQWHP AAQPAMAHHHHHH 100  97 9.4D VKKLL SLVYFFPFYP AAQPAMAHHHHHH 101  98 10.2H VKKLL FAFAPAPFYH AAQPAMAHHHHHH 102  99 4.12B VKKLL FLPFALVPRQ AAQPAMAHHHHHH 103 100 4.1F VKKLL ALWMqLYPQD AAQPAMAHHHHHH 104 101 YJ2.27 VKKLL ASILFSHAAP AAQPAMAHHHHHH 105 102 2.2C VKKLL LPLPWSLHLY AAQPAMAHHHHHH 106 103 4.9C VKKLL LPHFMSFWFE AAQPAMAHHHHHH 107 104 7.3E VKKLL LFQPFWPIPY AAQPAMAHHHHHH 108 105 4.7F VKKLL LLFSLGRLPP AAQPAMAHHHHHH 109 106 7.12G VKKLL PLWVLLKDPL AAQPAMAHHHHHH 110 107 9.3B VKKLL MSFATLFPHN AAQPAMAHHHHHH 111 108 4.5B VKKLL qHSLVTSWLC AAQPAMAHHHHHH 112 109 5.2H VKKLL LLFqGAFVGq AAQPAMAHHHHHH 113 110 4.4C VKKLL WMFHSLPFSP AAQPAMAHHHHHH 114 111 6.8G VKKLL LTqLLLTRLH AAQPAMAHHHHHH 115 112 4.10A VKKLL ALTLVPSSYP AAQPAMAHHHHHH 116 113 4.5D VKKLL LPWYMLLSDS AAQPAMAHHHHHH 117 114 9.3E VKKLL VVTqFWPSLP AAQPAMAHHHHHH 118 115 4.3G VKKLL LSTLFLWHVR AAQPAMAHHHHHH 119 116 9.7E VKKLL RSLFFqqLYP AAQPAMAHHHHHH 120 117 YJ2.30 VKKLL TLTTLHQTFP AAQPAMAHHHHHH 121 118 1.3B VKKLL SALLAPWYWD AAQPAMAHHHHHH 122 119 8.9B VKKLL AIqqRMQIYT AAQPAMAHHHHHH 123 120 3.4E VKKLL LLFPWFQPPY AAQPAMAHHHHHH 124 121 9.23.7E VKKLL YFTSLLGqFP AAQPAMAHHHHHH 125 122 6.3D VKKLL PVLIFLSEIR AAQPAMAHHHHHH 126 123 9.5G VKKLL VATSLRWAVT AAQPAMAHHHHHH 127 124 YJ2.54 VKKLL AQLFHLFATH AAQPAMAHHHHHH 128 125 8.6G VKKLL LqFSALFNSF AAQPAMAHHHHHH 129 126 7.12C-q VKKLL FHLMSMLPPP AAQPAMAHHHHHH 130 127 5.4C VKKLL PVCSqSMFPI AAQPAMAHHHHHH 131 128 YJ2.48 VKKLL LLLSSSYQSP AAQPAMAHHHHHH 132 129 4.3D VKKLL LDSLFFHAPL AAQPAMAHHHHHH 133 130 7.7A VKKLL qAWVFSAHQL AAQPAMAHHHHHH 134 131 YJ2.99 VKKLL FQALGALTSP AAQPAMAHHHHHH 135 132 9.9D VKKLL CFFFFLqFHP AAQPAMAHHHHHH 136 133 4.12F-f VKKLL CFSHLALPSP AAQPAMAHHHHHH 137 134 6.2B VKKLL FGSWIPFTQM AAQPAMAHHHHHH 138 135 4.6F VKKLL GLGYFNWTLL AAQPAMAHHHHHH 139 136 10.4A VKKLL HLFPLFQFHH AAQPAMAHHHHHH 140 137 5.6B VKKLL SEHVSSICVL AAQPAMAHHHHHH 141 138 3.11E VKKLL FSCLLDPTCP AAQPAMAHHHHHH 142 139 8.3F VKKLL LYLLHPSFLP AAQPAMAHHHHHH 143 140 2.2F VKKLL WCAPLLYSLR AAQPAMAHHHHHH 144 141 2.3F VKKLL FAMFPYTFqT AAQPAMAHHHHHH 145 142 10.5D VKKLL LPSLFYVESL AAQPAMAHHHHHH 146 143 8.8B VKKLL SLWLSSLSVL AAQPAMAHHHHHH 147 144 YJ2.17 VKKLL PHLWFLWSLK AAQPAMAHHHHHH 148 145 7.5B VKKLL ASDPVWYFLW AAQPAMAHHHHHH 149 146 10.12D VKKLL GLPLMGLqSL AAQPAMAHHHHHH 150 147 2.4H VKKLL PQLLLLRALS AAQPAMAHHHHHH 151 148 5.5D VKKLL APSAFSLHLF AAQPAMAHHHHHH 152 149 9.4C VKKLL FqLSSLFVPY AAQPAMAHHHHHH 153 150 4.5H VKKLL VPSFLSTMIE AAQPAMAHHHHHH 154 151 2.7B VKKLL ASPFFASYLW AAQPAMAHHHHHH 155 152 YJ2.23 VKKLL LQYLLSPIGY AAQPAMAHHHHHH 156 153 6.2D VKKLL VLSVPISAHH AAQPAMAHHHHHH 157 154 7.4A VKKLL MMqALSSLPE AAQPAMAHHHHHH 158 155 4.12B VKKLL MPAVLATRLT AAQPAMAHHHHHH 159 156 6.12E VKKLL PFTAWIIDGW AAQPAMAHHHHHH 160 157 YJ2.125 VKKLL TQLLPLWQPL AAQPAMAHHHHHH 161 158 YJ2.21 VKKLL LVPSLLPLTQ AAQPAMAHHHHHH 162 159 10.12B VKKLL PIqSCMVIPS AAQPAMAHHHHHH 163 160 YJ2.35 VKKLL WSLHLATRLL AAQPAMAHHHHHH 164 161 6.11H VKKLL qQVLLCSTLR AAQPAMAHHHHHH 165 162 7.3B VKKLL LLRYFLDPMY AAQPAMAHHHHHH 166 163 10.12A VKKLL IPQFLRSHHR AAQPAMAHHHHHH 167 164 YJ2.6 VKKLL GVLHLALSLR AAQPAMAHHHHHH 168 165 4.12C VKKLL LVTSqFSLVP AAQPAMAHHHHHH 169 166 YJ2.19 VKKLL PLALSWFQLR AAQPAMAHHHHHH 170 167 YJ2.88 VKKLL QHQWYPTVLM AAQPAMAHHHHHH 171 168 YJ2.29 VKKLL LMYWLSKPLS AAQPAMAHHHHHH 172 169 YJ2.8 VKKLL TQLTLSSSPI AAQPAMAHHHHHH 173 170 YJ2.94 VKKLL QLTALLSRLI AAQPAMAHHHHHH 174 171 YJ2.107 VKKLL LMTFGTTPQS AAQPAMAHHHHHH 175 172 YJ2.133 VKKLL SAFSFSLSST AAQPAMAHHHHHH 176 173 6.1A VKKLL APWLVLPHFP AAQPAMAHHHHHH 177 174 YJ2.81 VKKLL HVLSFAPPMP AAQPAMAHHHHHH 178 175 YJ2.38 VKKLL NWLFFAHPFS AAQPAMAHHHHHH 179 176 YJ2.20 VKKLL QLAVLLGSLR AAQPAMAHHHHHH 180 177 7.1D VKKLL LFGLFYFRAC AAQPAMAHHHHHH 181 178 YJ2.98 VKKLL FQFFVVWRLL AAQPAMAHHHHHH 182 179 YJ2.39 VKKLL PWAWPPPPFW AAQPAMAHHHHHH 183 180 YJ2.130 VKKLL LQLVIVYYLR AAQPAMAHHHHHH 184 181 YJ2.16 VKKLL RQSVLLSALH AAQPAMAHHHHHH 185 182 3.12E VKKLL VYGYFLTTFR AAQPAMAHHHHHH 186 183 YJ2.53 VKKLL CFSPLFGFHT AAQPAMAHHHHHH 187 184 YJ2.100 VKKLL PGYALWQTI PAAQPAMAHHHHHH 188 185 YJ2.58 VKKLL QRIFICFFLR AAQPAMAHHHHHH 189 186 8.2A VKKLL PHVFSCqLSA AAQPAMAHHHHHH 190 187 5.10A VKKLL SPLSLSVKLL AAQPAMAHHHHHH 191 188 9.2D VKKLL ARSLFSGSML AAQPAMAHHHHHH 192 189 YJ2.92 VKKLL LQFLIVFPLR AAQPAMAHHHHHH 193 190 YJ2.32 VKKLL LAVLLGQSLR AAQPAMAHHHHHH 194 191 YJ2.14 VKKLL LLSHLFLRLH AAQPAMAHHHHHH 195 192 8.4E VKKLL LAMVFFVTLR AAQPAMAHHHHHH 196 193 YJ2.117 VKKLL WLFALPQENV AAQPAMAHHHHHH 197 194 YJ2.66 VKKLL HPLVLLSSSP AAQPAMAHHHHHH 198 195 YJ2.131 VKKLL LQYLFMLSMR AAQPAMAHHHHHH 199 196 4.11H VKKLL PALLIRYASV AAQPAMAHHHHHH 200 197 YJ2.78 VKKLL QQFTSPFLLL AAQPAMAHHHHHH 201 198 YJ2.44 VKKLL SPCFFLLYLR AAQPAMAHHHHHH 202 199 YJ2.90 VKKLL PGMPLFFTNS AAQPAMAHHHHHH 203 200 YJ2.47 VKKLL PQVFFLFRPF AAQPAMAHHHHHH 204 201 YJ2.110 VKKLL PFPILLQSPF AAQPAMAHHHHHH 205 202 YJ2.74 VKKLL FQACCLFPLQ AAQPAMAHHHHHH 206 203 YJ2.55 VKKLL AVVHTMPLFS AAQPAMAHHHHHH 207 204 YJ2.108 VKKLL QFSWAFVSIL AAQPAMAHHHHHH 208 205 YJ2.96 VKKLL PVCLFWSFFR AAQPAMAHHHHHH 209 206 YJ2.70 VKKLL QLLWQQQVPV AAQPAMAHHHHHH 210 207 YJ2.60 VKKLL PLQALSWFLR AAQPAMAHHHHHH 211 208 YJ2.119 VKKLL FYLLCRLSLQ AAQPAMAHHHHHH 212 209 YJ2.82 VKKLL YLQILVICLR AAQPAMAHHHHHH 213 210 YJ2.63 VKKLL QLFLIVFPLR AAQPAMAHHHHHH 214 211 10.5A VKKLL PLHFALFFRL AAQPAMAHHHHHH 215 212 YJ2.85 VKKLL PFPMHLVLPF AAQPAMAHHHHHH 216 213 YJ2.86 VKKLL PLLFSPPSLH AAQPAMAHHHHHH 217 214 YJ2.126 VKKLL CQSITFSSIW AAQPAMAHHHHHH 218 215 YJ2.112 VKKLL WQRLFPFLLI AAQPAMAHHHHHH 219 216 YJ2.77 VKKLL MVPFWPFSFT AAQPAMAHHHHHH 220 217 YJ2.103 VKKLL QAFPLPPLLV AAQPAMAHHHHHH 221 218 YJ2.134 VKKLL PLYLLFRSFV AAQPAMAHHHHHH 222 219 YJ2.91 VKKLL HRSMYLSWLY AAQPAMAHHHHHH 223 220 YJ2.64 VKKLL LLSTLVRAPY AAQPAMAHHHHHH 224 221 YJ2.87 VKKLL PLALSQWFLR AAQPAMAHHHHHH 225 222 YJ2.116 VKKLL AQGMIFFLRL AAQPAMAHHHHHH 226 223 YJ2.62 VKKLL FCCRLALQFF AAQPAMAHHHHHH 227 224 YJ2.102 VKKLL YLQFLSLMLS AAQPAMAHHHHHH 228 225 YJ2.106 VKKLL CQATFPTLLC AAQPAMAHHHHHH 229 226 YJ2.124 VKKLL ARSYLYFSLS AAQPAMAHHHHHH 230 227 YJ2.111 VKKLL YQSSFLPLFW AAQPAMAHHHHHH 231 228 YJ2.104 VKKLL SASFLAFRIT AAQPAMAHHHHHH 232 229 YJ2.67 VKKLL SVLFLSHYHS AAQPAMAHHHHHH 233 230 YJ2.105 VKKLL PLALLYVRLS AAQPAMAHHHHHH 234 231 YJ2.127 VKKLL PEFLLLFRFF AAQPAMAHHHHHH 235 232 YJ2.80 VKKLL FPSLYAWGGL AAQPAMAHHHHHH 236 233 YJ2.122 VKKLL LQAAAFFCWL AAQPAMAHHHHHH 237 234 YJ2.79 VKKLL PFFLFCSSLR AAQPAMAHHHHHH 238 235 YJ2.115 VKKLL ELTQLWLFHL AAQPAMAHHHHHH 239 236 YJ2.113 VKKLL PGVPLLLCFR AAQPAMAHHHHHH 240 237 YJ2.114 VKKLL SQAYLSYFLY AAQPAMAHHHHHH 241 238 YJ2.61 VKKLL ISYAFLVRVT AAQPAMAHHHHHH 242 239 YJ2.123 VKKLL APALLRSILA AAQPAMAHHHHHH 243 240 YJ2.109 VKKLL HSHTLLMSLH AAQPAMAHHHHHH 244 241 YJ2.83 VKKLL AVSAFVSLVR AAQPAMAHHHHHH 245 242 YJ2.31 VKKLL TLITFKFLPH AAQPAMAHHHHHH 246 243 YJ2.49 VKKLL QQFAIPLVEF AAQPAMAHHHHHH 247 244 YJ2.75 VKKLL MPCLLVYYLE AAQPAMAHHHHHH 248 245 YJ2.71 VKKLL RYCLLLQIVR AAQPAMAHHHHHH 249 246 YJ2.45 VKKLL SLALLRVSLG AAQPAMAHHHHHH 250 247 YJ2.68 VKKLL IIGRIALILR AAQPAMAHHHHHH 251 248 YJ2.24 VKKLL PQLICAFILR AAQPAMAHHHHHH 252 249 8.3E VKKLL MVPLFPLPLP AAQPAMAHHHHHH 253 250 8.1B VKKLL HqAILYYYLN AAQPAMAHHHHHH 254

TABLE 3 Preference sequence patterns selected from L3 S5 sc-dsFv library No. Code Sequence SEQ ID NO M13-pe1B VKKLLFAIPL VVPFYAAQPA MAHHHHHH   4 1 2.1A VKKLLFAIPL LPAQAMPMSR MAHHHHHH * 255 2 7.5C VKKLLFAIPL YFVLVRESSS MAHHHHHH * 256 3 1.3B VKKLLFAIPL VLVVSSRTRA MAHHHHHH * 257 4 YJ3.25 VKKLLFAIPL LLSRPRAVPD MAHHHHHH * 258 5 3.8A VKKLLFAIPL CVSVRSPAFA MAHHHHHH * 259 6 1.6A VKKLLFAIPL MTTLASRTHA MAHHHHHH * 260 7 1.4H VKKLLFAIPL YLSMTRSGAA MAHHHHHH * 261 8 7.8F VKKLLFAIPL WLRSSVPVDS MAHHHHHH * 262 9 7.8H VKKLLFAIPL LSSLTRDSSS MAHHHHHH * 263 10 7.5E VKKLLFAIPL GLFTIRDSFA MAHHHHHH * 264 11 7.6C VKKLLFAIPL WLGITKPVWS MAHHHHHH * 265 12 1.3F VKKLLFAIPL YTLTPRPVFS MAHHHHHH * 266 13 1.5F VKKLLFAIPL gLALSRPSFP MAHHHHHH * 267 14 14.9A VKKLLFAIPL SSFLVADQSS MAHHHHHH * 268 15 YJ3.7 VKKLLFAIPL LLGLASPRSR MAHHHHHH * 269 16 13.1E VKKLLFAIPL LTLSNRSAWS MAHHHHHH * 270 17 2.2C VKKLLFAIPL LSLYPTRSTA MAHHHHHH * 271 18 YJ3.10 VKKLLFAIPL LTTLSRPSFS MAHHHHHH * 272 19 8.1A VKKLLFAIPL YFSRPPqPSS MAHHHHHH * 273 20 6.2H VKKLLFAIPL TMSSPPRSTS MAHHHHHH * 274 21 8.1C VKKLLFAIPL YFLRISPSAS MAHHHHHH * 275 22 1.8B VKKLLFAIPL LFLRPSAARP MAHHHHHH * 276 23 1.8C VKKLLFAIPL LWSSSRPTSQ MAHHHHHH * 277 24 YJ3.41 VKKLLFAIPL YLVCSRPLHA MAHHHHHH * 278 25 10.8G VKKLLFAIPL VLQRPPSPNT MAHHHHHH * 279 26 2.7C VKKLLFAIPL AMASFRPRDQ MAHHHHHH * 280 27 7.10C VKKLLFAIPL SRSLAMQPLP MAHHHHHH * 281 28 1.2A VKKLLFAIPL LSSLRSSNPE MAHHHHHH * 282 29 YJ3.4 VKKLLFAIPL SILINFRASS MAHHHHHH * 283 30 1.6B VKKLLFAIPL YWRSFWEPPA MAHHHHHH * 284 31 4.8E VKKLLFAIPL YLAAPRSTVA MAHHHHHH * 285 32 6.7H VKKLLFAIPL QYSAFSMSPR MAHHHHHH * 286 33 7.9C VKKLLFAIPL YLVSSKNSYP MAHHHHHH * 287 34 YJ3.72 VKKLLFAIPL GLSVSFRTSA MAHHHHHH * 288 35 4.4C VKKLLFAIPL AMLEPTRSSA MAHHHHHH * 289 36 11.1B VKKLLFAIPL SLSLHRPALA MAHHHHHH * 290 37 6.6B VKKLLFAIPL LSASARGSYA MAHHHHHH * 291 38 YJ3.26 VKKLLFAIPL YLAVTHRAYS MAHHHHHH * 292 39 YJ3.44 VKKLLFAIPL FFSLSRYSLA MAHHHHHH * 293 40 5.4B VKKLLFAIPL YLSAPRHASP MAHHHHHH * 294 41 5.2D VKKLLFAIPL WSFSRLPSSD MAHHHHHH * 295 42 12.4E VKKLLFAIPL YLSLTKPSLS MAHHHHHH * 296 43 14.1C VKKLLFAIPL SSPATEVLSP MAHHHHHH * 297 44 6.2C VKKLLFAIPL TLFLQRSSLA MAHHHHHH * 298 45 YJ3.6 VKKLLFAIPL VFTRVPHKPS MAHHHHHH * 299 46 4.1E VKKLLFAIPL AITRSSQFPS MAHHHHHH * 300 47 6.4H VKKLLFAIPL LGDLRSSPDA MAHHHHHH * 301 48 YJ3.53 VKKLLFAIPL VTTLSTRCYA MAHHHHHH * 302 49 7.7B VKKLLFAIPL FDASLEGPAM MAHHHHHH * 303 50 11.3C VKKLLFAIPL YFSSPSSRAP MAHHHHHH * 304 51 1.12A VKKLLFAIPL WFSFPFRSAA MAHHHHHH 305 52 12.1A VKKLLFAIPL YLSMSSPARS MAHHHHHH 306 53 1.12D VKKLLFAIPL SWSLCRPVCA MAHHHHHH 307 54 4.3G VKKLLFAIPL LYCWPRHSWS MAHHHHHH 308 55 YJ3.38 VKKLLFAIPL IFYTTRSSLS MAHHHHHH 309 56 YJ3.45 VKKLLFAIPL IYTLRSHSMT MAHHHHHH 310 57 2.9H VKKLLFAIPL PVPSLLGSAD MAHHHHHH 311 58 9.5A VKKLLFAIPL SLSLNSRSYP MAHHHHHH 312 59 2.7H VKKLLFAIPL FSPTSQEIRH MAHHHHHH 313 60 2.2G VKKLLFAIPL YFSCPLRVAS MAHHHHHH 314 61 YJ3.81 VKKLLFAIPL VLSLNRGVFA MAHHHHHH 315 62 7.4H VKKLLFAIPL SPqVLSSSPG MAHHHHHH 316 63 4.2C VKKLLFAIPL YVNAMSSPRP MAHHHHHH 317 64 13.6D VKKLLFAIPL YFTFVRSSWC MAHHHHHH 318 65 5.8D VKKLLFAIPL FDLSSDSVSP MAHHHHHH 319 66 YJ3.47 VKKLLFAIPL YILFWRNTHA MAHHHHHH 320 67 13.7A VKKLLFAIPL SCFLSRSAFS MAHHHHHH 321 68 YJ3.83 VKKLLFAIPL FFMITSKSRS MAHHHHHH 322 69 12.6C VKKLLFAIPL IVSSSRGSFA MAHHHHHH 323 70 4.10B VKKLLFAIPL AASRPLSPAA MAHHHHHH 324 71 YJ3.46 VKKLLFAIPL WLFSPLRSYS MAHHHHHH 325 72 YJ3.56 VKKLLFAIPL FLSYVRPLSA MAHHHHHH 326 73 13.5G VKKLLFAIPL FIFTPRSVHS MAHHHHHH 327 74 2.2E VKKLLFAIPL VSSIYKNSPP MAHHHHHH 328 75 5.5H VKKLLFAIPL MSDSTAPSFA MAHHHHHH 329 76 6.4B VKKLLFAIPL TLPqPRFPSP MAHHHHHH 330 77 7.10G VKKLLFAIPL SLLADSPRRP MAHHHHHH 331 78 5.3A VKKLLFAIPL FTDNSGEPSL MAHHHHHH 332 79 11.1E VKKLLFAIPL YCMPMSRTCA MAHHHHHH 333 80 11.1D VKKLLFAIPL MSRLSYHTPS MAHHHHHH 334 81 2.2F VKKLLFAIPL LSNSRVPPSS MAHHHHHH 335 82 15.7A VKKLLFAIPL FFASMRHTqA MAHHHHHH 336 83 YJ3.5 VKKLLFAIPL LLSTIKTSFS MAHHHHHH 337 84 3.3A VKKLLFAIPL FQQSSLSSVP MAHHHHHH 338 85 16.11A VKKLLFAIPL TLILSHRSSA MAHHHHHH 339 86 11.12A VKKLLFAIPL SFSRDPSFTS MAHHHHHH 340 87 9.1B VKKLLFAIPL ALSPTRHTLA MAHHHHHH 341 88 13.9A VKKLLFAIPL NILFTVRVYA MAHHHHHH 342 89 YJ3.15 VKKLLFAIPL LASLSARCHG MAHHHHHH 343 90 12.6B VKKLLFAIPL SVTLSLRASA MAHHHHHH 344 91 15.8H VKKLLFAIPL SHDPLLLSSP MAHHHHHH 345 92 YJ3.71 VKKLLFAIPL LWSLSSRGMT MAHHHHHH 346 93 YJ3.82 VKKLLFAIPL LISYCRPVSS MAHHHHHH 347 94 9.1D VKKLLFAIPL HSVELPASPA MAHHHHHH 348 95 9.6A VKKLLFAIPL LLSTSRSSSG MAHHHHHH 349 96 YJ3.34 VKKLLFAIPL WFSCSRFALS MAHHHHHH 350 97 YJ3.28 VKKLLFAIPL VCTLSSRAFS MAHHHHHH 351 98 11.1H VKKLLFAIPL YSPLARNPFS MAHHHHHH 352 99 16.9D VKKLLFAIPL FFAFSRQSSG MAHHHHHH 353 100 YJ3.70 VKKLLFAIPL TFSIFSRALA MAHHHHHH 354 101 YJ3.55 VKKLLFAIPL SLFFSARAIA MAHHHHHH 355 102 9.7A VKKLLFAIPL SQPSLCDPVP MAHHHHHH 356 103 10.11A VKKLLFAIPL LASYHRVAFA MAHHHHHH 357 104 10.1F VKKLLFAIPL WQLWQLPSRP MAHHHHHH 358 105 16.8A VKKLLFAIPL FTPMYRPTSP MAHHHHHH 359 106 YJ3.27 VKKLLFAIPL LLSLHRFSFA MAHHHHHH 360 107 9.5H VKKLLFAIPL SYSHPQNALA MAHHHHHH 361 108 10.12D VKKLLFAIPL YVLRSDASWG MAHHHHHH 362 109 4.2D VKKLLFAIPL FSGPPFDRTS MAHHHHHH 363 110 YJ3.66 VKKLLFAIPL FCALSRFTHA MAHHHHHH 364 111 YJ3.24 VKKLLFAIPL FSLSRPVPPL MAHHHHHH 365 112 10.7D VKKLLFAIPL SMDSFSRPFF MAHHHHHH 366 113 15.7C VKKLLFAIPL YTIIPSRASS MAHHHHHH 367 114 15.12C VKKLLFAIPL VPSANPPPLS MAHHHHHH 368 115 15.7E VKKLLFAIPL YLIKPPEGFS MAHHHHHH 369 116 YJ3.42 VKKLLFAIPL ISTLHFRAFG MAHHHHHH 370 117 YJ3.37 VKKLLFAIPL VRVMCGHSYA MAHHHHHH 371 118 YJ3.67 VKKLLFAIPL VLSLSRTFSG MAHHHHHH 372 119 YJ3.75 VKKLLFAIPL WCALSRQSMP MAHHHHHH 373 120 YJ3.86 VKKLLFAIPL YFWSLRVSWP MAHHHHHH 374 121 YJ3.33 VKKLLFAIPL YILSPRLPPP MAHHHHHH 375 122 YJ3.22 VKKLLFAIPL VVAAHRFSYA MAHHHHHH 376 123 YJ3.62 VKKLLFAIPL YVHLTSKAIP MAHHHHHH 377 124 YJ3.59 VKKLLFAIPL SLTLYRSGWS MAHHHHHH 378 125 YJ3.18 VKKLLFAIPL YYALSGRPVT MAHHHHHH 379 126 YJ3.79 VKKLLFAIPL MLSLMRQSAP MAHHHHHH 380

TABLE 4 Preference sequence patterns selected from L4 S5 sc-dsFv library No. Code Sequence SEQ ID NO M13-pe1B VKKLLFAIPLVVPFY AAQPAMAHHH HHH   4 1 1.11A VKKLLFAIPLVVPFY ARPLTRIQTP HHH * 381 2 9.3D VKKLLFAIPLVVPFY LTQLSRREPS HHH * 382 3 1.6B VKKLLFAIPLVVPFY ARSLATSPSR HHH * 383 4 14.5H VKKLLFAIPLVVPFY PARSYMLVRP HHH * 384 5 12.2A VKKLLFAIPLVVPFY SRSYMLLSRP HHH * 385 6 12.6H VKKLLFAIPLVVPFY TRSALAFFLP HHH * 386 7 YJ4.13 VKKLLFAIPLVVPFY SRGFTLPRLI HHH * 387 8 YJ4.1 VKKLLFAIPLVVPFY SSAFTRPIRP HHH * 388 9 12.2E VKKLLFAIPLVVPFY TRYSHAFMLI HHH * 389 10 6.10B VKKLLFAIPLVVPFY ARPMSMFRSD HHH * 390 11 8.4D VKKLLFAIPLVVPFY ASSMSqYRQN HHH * 391 12 5.9H VKKLLFAIPLVVPFY ARSYSRPPSI HHH * 392 13 10.8A VKKLLFAIPLVVPFY ASSMSRLRPH HHH * 393 14 YJ4.3 VKKLLFAIPLVVPFY CRSLSRPMLV HHH * 394 15 4.6C VKKLLFAIPLVVPFY SRSMSLHPTA HHH * 395 16 CM11 VKKLLFAIPLVVPFY TRSMTRLAPP HHH * 396 17 9.8H VKKLLFAIPLVVPFY TRAMSVSHKT HHH * 397 18 13.1F VKKLLFAIPLVVPFY LLAPKPSVKR HHH * 398 19 9.7A VKKLLFAIPLVVPFY SRPAPALSRL HHH * 399 20 15.9C VKKLLFAIPLVVPFY AKAMSARYQS HHH * 400 21 CM18 VKKLLFAIPLVVPFY FASQRSSPIR HHH * 401 22 CM24 VKKLLFAIPLVVPFY CLSFTSARFq HHH * 402 23 12.1A VKKLLFAIPLVVPFY PSASSRLSPK HHH * 403 24 2.10G VKKLLFAIPLVVPFY ARSYTRVPLA HHH * 404 25 CM2 VKKLLFAIPLVVPFY ARSLTFLPPR HHH * 405 26 9.4C VKKLLFAIPLVVPFY TTRVNAFMLV HHH * 406 27 11.11H VKKLLFAIPLVVPFY QAFRPVPVRN HHH * 407 28 11.8H VKKLLFAIPLVVPFY TSGMSRLRSW HHH * 408 29 1.12C VKKLLFAIPLVVPFY SRSPSQLSSR HHH * 409 30 16.12H VKKLLFAIPLVVPFY AFSLSRTSSK HHH * 410 31 3.11F VKKLLFAIPLVVPFY FHRVQQFSPA HHH * 411 32 9.2B VKKLLFAIPLVVPFY LDSMLTFRRS HHH * 412 33 CM40 VKKLLFAIPLVVPFY CRSLTSPLRM HHH * 413 34 15.5B VKKLLFAIPLVVPFY SRSASFLRPI HHH * 414 35 9.2F VKKLLFAIPLVVPFY MTFqSNSPRG HHH * 415 36 CM38 VKKLLFAIPLVVPFY CRPMTLRqPV HHH * 416 37 CM5 VKKLLFAIPLVVPFY VRPMSRVIMS HHH * 417 38 CM36 VKKLLFAIPLVVPFY SYGFSRPFSK HHH * 418 39 11.9G VKKLLFAIPLVVPFY TRSCFAFMLP HHH * 419 40 6.8B VKKLLFAIPLVVPFY AFSGAFRQSQ HHH * 420 41 16.6B VKKLLFAIPLVVPFY LRAGSFSAAP HHH * 421 42 CM22 VKKLLFAIPLVVPFY SHSMAPPSRR HHH * 422 43 CM31 VKKLLFAIPLVVPFY CRSGTFGNIG HHH * 423 44 11.5F VKKLLFAIPLVVPFY ARSMASTPLA HHH * 424 45 YJ4.2 VKKLLFAIPLVVPFY VYPLAPRLRD HHH * 425 46 6.10H VKKLLFAIPLVVPFY SLPWRRTPFQ HHH * 426 47 10.3D VKKLLFAIPLVVPFY MRTPPLSqRI HHH * 427 48 CM28 VKKLLFAIPLVVPFY ARSLSSYNAV HHH * 428 49 12.4D VKKLLFAIPLVVPFY VHALARKSQF HHH * 429 50 CM25 VKKLLFAIPLVVPFY SRSFSSPSIT HHH 430 51 13.5A VKKLLFAIPLVVPFY CRALSKPLPP HHH 431 52 12.6C VKKLLFAIPLVVPFY CRPSAPKMLL HHH 432 53 CM16 VKKLLFAIPLVVPFY SRSMSYFqPL HHH 433 54 4.2C VKKLLFAIPLVVPFY TRSLSRSIPH HHH 434 55 16.6C VKKLLFAIPLVVPFY SQLHqSPGNP HHH 435 56 10.10A VKKLLFAIPLVVPFY TRAIARPPYT HHH 436 57 10.11G VKKLLFAIPLVVPFY ARSLSTVRFP HHH 437 58 CM8 VKKLLFAIPLVVPFY TRAFSSPLSN HHH 438 59 9.6D VKKLLFAIPLVVPFY NRTPTIqRDS HHH 439 60 8.4B VKKLLFAIPLVVPFY ARAVSRTVPT HHH 440 61 8.5E VKKLLFAIPLVVPFY AqSMAVPIST HHH 441 62 13.2C VKKLLFAIPLVVPFY PqPSRGFMLI HHH 442 63 CM10 VKKLLFAIPLVVPFY TRSMVFPAKV HHH 443 64 CM26 VKKLLFAIPLVVPFY SRSMTLKGPE HHH 444 65 CM17 VKKLLFAIPLVVPFY AFPFSRQPNA HHH 445 66 CM7 VKKLLFAIPLVVPFY SRALTSISGM HHH 446 67 CM6 VKKLLFAIPLVVPFY CRGMSLNVTR HHH 447 68 6.10C VKKLLFAIPLVVPFY SHWRTQRPPE HHH 448 69 CM45 VKKLLFAIPLVVPFY ARSFSSPPGP HHH 449 70 13.1G VKKLLFAIPLVVPFY IFPIEASARR HHH 450 71 CM39 VKKLLFAIPLVVPFY ASSMALRPRV HHH 451 72 YJ4.74 VKKLLFAIPLVVPFY SRAFSSTPAM HHH 452 73 1.7F VKKLLFAIPLVVPFY SRSMVLQGPT HHH 453 74 YJ4.28 VKKLLFAIPLVVPFY SRSMTSPPYI HHH 454 75 10.3B VKKLLFAIPLVVPFY ANRPQSTKNI HHH 455 76 YJ4.56 VKKLLFAIPLVVPFY SRALTMTPSF HHH 456 77 4.6H VKKLLFAIPLVVPFY PTRLFAFMLT HHH 457 78 14.12 AVKKLLFAIPLVVPF YSRAMSPIPR HHH 458 79 CM29 VKKLLFAIPLVVPFY ARSMGSMWQL HHH 459 80 YJ4.42 VKKLLFAIPLVVPFY SFSMTRSSPL HHH 460 81 CM42 VKKLLFAIPLVVPFY SFSFIRqPLP HHH 461 82 YJ4.33 VKKLLFAIPLVVPFY NRVPSPASQT HHH 462 83 YJ4.23 VKKLLFAIPLVVPFY SFSFSKPRFS HHH 463 84 CM27 VKKLLFAIPLVVPFY ARSLTQFSSV HHH 464 85 YJ4.39 VKKLLFAIPLVVPFY ARCFSSPVAL HHH 465 86 11.3B VKKLLFAIPLVVPFY GASSWWLFPS HHH 466 87 YJ4.84 VKKLLFAIPLVVPFY TPPQQQALLS HHH 467 88 14.1F VKKLLFAIPLVVPFY SRGFSMAFFP HHH 468 89 CM33 VKKLLFAIPLVVPFY SLAMSRPqAS HHH 469 90 13.12C VKKLLFAIPLVVPFY TYALTTFqSV HHH 470 91 YJ4.44 VKKLLFAIPLVVPFY QHAFTRPFRV HHH 471 92 CM30 VKKLLFAIPLVVPFY SRAFSSPSGS HHH 472 93 13.11G VKKLLFAIPLVVPFY TSALARSPRV HHH 473 94 4.8B VKKLLFAIPLVVPFY CRAMSSPFRP HHH 474 95 4.2B VKKLLFAIPLVVPFY STFARSFMLT HHH 475 96 9.2D VKKLLFAIPLVVPFY FPLSSRAFML HHH * 476 97 YJ4.71 VKKLLFAIPLVVPFY SRSMSTSPIL HHH 477 98 9.6H VKKLLFAIPLVVPFY SFGLqLPqPF HHH 478 99 CM37 VKKLLFAIPLVVPFY SRSMSLSSDL HHH 479 100 16.3E VKKLLFAIPLVVPFY AFPLARRPIN HHH 480 101 12.1B VKKLLFAIPLVVPFY TSCRAMTLPR HHH 481 102 CM23 VKKLLFAIPLVVPFY TYPFSRAGPP HHH 482 103 YJ4.47 VKKLLFAIPLVVPFY ANQQALPFQL HHH 483 104 YJ4.38 VKKLLFAIPLVVPFY GWSMSLRSHS HHH 484 105 4.11H VKKLLFAIPLVVPFY SPQVVTRKDL HHH 485 106 12.9G VKKLLFAIPLVVPFY LRNAHAMASA HHH 486 107 CM44 VKKLLFAIPLVVPFY SRSGSFNVTP HHH 487 108 11.3E VKKLLFAIPLVVPFY SRPLSRVPVF HHH 488 109 11.9F VKKLLFAIPLVVPFY SKRMPPPISq HHH 489 110 CM34 VKKLLFAIPLVVPFY TRSMSSLPSP HHH 490 111 14.11D VKKLLFAIPLVVPFY CRSSSSIFPL HHH 491 112 CM15 VKKLLFAIPLVVPFY RSAHAMSIQT HHH 492 113 10.1H VKKLLFAIPLVVPFY GYCFSARIIR HHH 493 114 9.10A VKKLLFAIPLVVPFY PHLSPLqPQq HHH 494 115 CM43 VKKLLFAIPLVVPFY SFSFSRFPGL HHH 495 116 YJ4.48 VKKLLFAIPLVVPFY SSSMSLRPQF HHH 496 117 11.11D VKKLLFAIPLVVPFY SSPRARPVPP HHH 497 118 CM46 VKKLLFAIPLVVPFY ARSLSALSPY HHH 498 119 12.5C VKKLLFAIPLVVPFY PVRqLHTNLR HHH 499 120 10.2F VKKLLFAIPLVVPFY PTTSTPYqSP HHH 500 121 CM21 VKKLLFAIPLVVPFY VNALTFLPSq HHH 501 122 CM41 VKKLLFAIPLVVPFY ARSLSSPLTL HHH 502 123 YJ4.25 VKKLLFAIPLVVPFY TRPPTVGLRQ HHH 503 124 CM14 VKKLLFAIPLVVPFY TRALSPMSWq HHH 504 125 YJ4.6 VKKLLFAIPLVVPFY VFPFSRPLLR HHH 505 126 CM1 VKKLLFAIPLVVPFY VPRCLSMSLG HHH 506 127 YJ4.87 VKKLLFAIPLVVPFY QQPSFHPISR HHH 507 128 CM32 VKKLLFAIPLVVPFY SKAFSSFqAS HHH 508 129 10.6H VKKLLFAIPLVVPFY GYSMSqSGLT HHH 509 130 YJ4.40 VKKLLFAIPLVVPFY AQALTTRGLA HHH 510 131 YJ4.26 VKKLLFAIPLVVPFY VKSLTRPAFL HHH 511 132 12.4F VKKLLFAIPLVVPFY AqSRLRVYPP HHH 512 133 4.5B VKKLLFAIPLVVPFY PAIGFMLLRY HHH 513 134 12.3D VKKLLFAIPLVVPFY SFGTLVRPRP HHH 514 135 CM3 VKKLLFAIPLVVPFY IRRPVDPVMP HHH 515 136 YJ4.19 VKKLLFAIPLVVPFY FPLRQTHRYP HHH 516 137 13.2H VKKLLFAIPLVVPFY THSMQRPTGR HHH 517 138 10.5D VKKLLFAIPLVVPFY RHTqLSSSTS HHH 518 139 15.10D VKKLLFAIPLVVPFY SCGFSRLSKA HHH 519 140 CM35 VKKLLFAIPLVVPFY SRSFSQLPHI HHH 520 141 YJ4.43 VKKLLFAIPLVVPFY SSSMSQLRPF HHH 521 142 10.2B VKKLLFAIPLVVPFY CRTTFALQSS HHH 522 143 CM19 VKKLLFAIPLVVPFY AQSMSIRHNN HHH 523 144 11.4E VKKLLFAIPLVVPFY NSRFRTTPPS HHH 524 145 CM20 VKKLLFAIPLVVPFY SVSMSRYQLS HHH 525 146 CM12 VKKLLFAIPLVVPFY SSGASRLRIL HHH 526 147 YJ4.81 VKKLLFAIPLVVPFY CWSLSRPRLL HHH 527 148 10.1C VKKLLFAIPLVVPFY TSRSTKLTPS HHH 528 149 11.6D VKKLLFAIPLVVPFY SRVSVAFMLM HHH 529 150 YJ4.72 VKKLLFAIPLVVPFY CLGRSMAPGP HHH 530 151 14.1A VKKLLFAIPLVVPFY FVHRRDSSSL HHH 531 152 YJ4.24 VKKLLFAIPLVVPFY SLGFSRLTSL HHH 532 153 13.2B VKKLLFAIPLVVPFY ASALSRRVPq HHH 533 154 11.6B VKKLLFAIPLVVPFY TYPASWPRLR HHH 534 155 9.2G VKKLLFAIPLVVPFY SRVSLAVTPS HHH 535 156 10.11B VKKLLFAIPLVVPFY NNPFSSLSqq HHH 536 157 11.8D VKKLLFAIPLVVPFY RPLPRPFAGN HHH 537 158 CM4 VKKLLFAIPLVVPFY GFSMTQYLPq HHH 538 159 YJ4.75 VKKLLFAIPLVVPFY SSALSRSFYP HHH 539 160 YJ4.61 VKKLLFAIPLVVPFY TQQRCFAMHI HHH 540 161 YJ4.85 VKKLLFAIPLVVPFY IKHFYNSRPS HHH 541 162 YJ4.51 VKKLLFAIPLVVPFY FTRLPKESSP HHH 542 163 9.6G VKKLLFAIPLVVPFY LPAQPRVTRT HHH 543 164 CM13 VKKLLFAIPLVVPFY LRSMTLNTST HHH 544 165 YJ4.35 VKKLLFAIPLVVPFY PDTFSYSSQD HHH 545 166 YJ4.41 VKKLLFAIPLVVPFY FRNPQLPSSA HHH 546 167 YJ4.50 VKKLLFAIPLVVPFY FRPDRTPPSS HHH 547 168 9.8C VKKLLFAIPLVVPFY qSHTILPLPA HHH 548 169 CM9 VKKLLFAIPLVVPFY SSAFqPMVSS HHH 549 170 9.7H VKKLLFAIPLVVPFY QSRRLPILPL HHH 550 171 YJ4.31 VKKLLFAIPLVVPFY GQAYLPAPQL HHH 551 172 9.11B VKKLLFAIPLVVPFY TSRPRETLFL HHH 552 173 9.3G VKKLLFAIPLVVPFY TAASVVRSRD HHH 553 174 10.5F VKKLLFAIPLVVPFY VRGAAPKFSV HHH 554 175 YJ4.14 VKKLLFAIPLVVPFY FRHQPASVST HHH 555 176 9.8B VKKLLFAIPLVVPFY PTNAIAFFLq HHH 556 177 YJ4.59 VKKLLFAIPLVVPFY LKSLRSDTPN HHH 557 178 YJ4.22 VKKLLFAIPLVVPFY IKRPLPLAPT HHH 558 179 11.11F VKKLLFAIPLVVPFY ASSSKSRFML HHH 559 180 YJ4.82 VKKLLFAIPLVVPFY PWKPRLLPPQ HHH 560 181 9.1H VKKLLFAIPLVVPFY SRGFMLTLRY HHH 561 182 9.8E VKKLLFAIPLVVPFY CKARGIMPVF HHH 562 183 YJ4.17 VKKLLFAIPLVVPFY ASLPRLTSQS HHH 563 184 11.2B VKKLLFAIPLVVPFY qSSAFSYMLS HHH 564 185 10.7A VKKLLFAIPLVVPFY SFSSQRFLRP HHH 565 186 9.7G VKKLLFAIPLVVPFY TSSNTSRRFP HHH 566 187 11.10B VKKLLFAIPLVVPFY NqTAATAPPR HHH 567 188 10.8G VKKLLFAIPLVVPFY GAPLSWRRSY HHH 568 189 9.10D VKKLLFAIPLVVPFY CRSVWCIPRP HHH 569 190 9.1C VKKLLFAIPLVVPFY AKACLRPLQT HHH 570 191 9.6F VKKLLFAIPLVVPFY CLASSHRHRP HHH 571 192 11.3H VKKLLFAIPLVVPFY LRADSLAPKS HHH 572 193 9.9F VKKLLFAIPLVVPFY SVPQFSGRSR HHH 573 194 YJ4.78 VKKLLFAIPLVVPFY VYPARFPAKT HHH 574 195 YJ4.21 VKKLLFAIPLVVPFY NFMLRHPQTF HHH 575 196 YJ4.32 VKKLLFAIPLVVPFY YVPRFPPKSA HHH 576 197 YJ4.86 VKKLLFAIPLVVPFY LSPMSRTRYV HHH 577 198 YJ4.66 VKKLLFAIPLVVPFY TYPLTKPYRP HHH 578 199 YJ4.83 VKKLLFAIPLVVPFY SSYWSHRKPP HHH 579 200 10.8C VKKLLFAIPLVVPFY SPRTFAFFLM HHH 580 201 11.1A VKKLLFAIPLVVPFY LGPGIRKKPA HHH 581 202 9.4E VKKLLFAIPLVVPFY TRLCVAKVAG HHH 582 203 11.2E VKKLLFAIPLVVPFY RSLPASGASR HHH 583 204 10.5E VKKLLFAIPLVVPFY ASPRVKSYSP HHH 584 205 9.10F VKKLLFAIPLVVPFY PSRTFAFYLV HHH 585 206 9.4H VKKLLFAIPLVVPFY qqEFAMAHHH HHH 586 207 11.8B VKKLLFAIPLVVPFY PqSSKAFFLN HHH 587 208 11.2F VKKLLFAIPLVVPFY VKALRGSYPT HHH 588 209 11.7F VKKLLFAIPLVVPFY TqPSqVRYML HHH 589 210 11.9C VKKLLFAIPLVVPFY SARGqHVRPP HHH 590 211 10.11C VKKLLFAIPLVVPFY STRCPGFFLq HHH 591 212 11.6E VKKLLFAIPLVVPFY CPSVFSRTPP HHH 592 213 11.3A VKKLLFAIPLVVPFY DASSWRHFLS HHH 593

Example 4 Production of sc-dsFv Against H5 of Influenza Virus and Microarray Test

As described above, scFvs (8a and 12a) and their disulfide forms (ds-8a and ds-12a, respectively) to various hemagglutins (HAs) from different serotypes of influenza virus were developed. As shown in FIG. 5, the results indicated that selected scFv phage clones against H5 of influenza virus could be introduced to sc-dsFv directly but had lower binding affinity as compared with original scFvs. These results also suggested that the binding affinity could be enhanced by sc-dsFv phage panning procedures with the signal sequences described above.

The 8aS5 protein could be concentrated to 6 mg/ml without precipitation. The array studies suggested that 4 ng/spot of ds-8a protein could detect ˜10⁷ viruses in solution by using 40 nm fluorescence beads. In conclusion, the signal sequence derived from sc-dsFv phage production against VEGF from monoclonal antibody could be applied for sc-dsFv phage production against hemagglutinin from natural antibody repertoire. The binding affinity could be enhanced by sc-dsFv phage panning procedures to produce sc-dsFv with high binding capacity and better stability than scFv for further applications.

Example 5 Soluble Non-Fusion sc-dsFv Expressed with Suppressor E. coli Strain

The signal sequences resulting in the successful expression of the displayed sc-dsFv on phage rescued from suppressor E. coli strain ER2738 were more likely to result in secretion of the soluble non-fusion anti-VEGF sc-dsFv in a culture medium. Signal sequence phage library L4 was selected for binding to immobilized VEGF and the VEGF-binding enriched phage variants were amplified for the next round of selection/amplification cycle. The selection/amplification cycle was repeated for four rounds. After each round of selection/amplification cycle, a random collection of 96 phage variants were picked from the amplified phage population. These phage variants were used to infect E. coli ER2738 and the soluble sc-dsFv was expressed in the overnight cultures, which were tested for binding to immobilized VEGF with ELISA.

These random collections of phage variants were also used to infect E. coli HB2151 for the same assay to determine the sc-dsFv secretion. The result showed that, with ER2738 as the host, 0%, 0%, 2%, and 14% of the phage variants from 1^(st), 2^(nd), 3^(rd), and 4^(th) round of selection/amplification cycle respectively secreted functional sc-dsFv binding to VEGF with ELISA signal greater than OD_(450 nm)>0.6. But this trend was not found in the experiment with E. coli strain HB2151. This result indicated that signal sequence alteration could restore the secretion of the soluble non-fusion sc-dsFv and that the search for the optimum signal sequences could be facilitated with phage-based selection/amplification cycles on signal sequence libraries. This conclusion is applicable only to the E. coli suppressor strain ER2738 as the bacteria host for the M13 phage.

Example 6 Interface Disulfide Bond Formation in the sc-dsFv

One measurement for the folding quality of the sc-dsFv is the extent of the interface disulfide bond formation in the sc-dsFv. This measurement was determined by the ratio of the sc-dsFv-VEGF binding ELISA signal after the fXa (bovine factor Xa) treatment over that before the fXa treatment. FXa cleaves substrate sequence -IEGR- in the linker peptide connecting the two variable domains in the sc-dsFv construct. If the interface disulfide bond was not formed in the sc-dsFv, the cleavage of the linker peptide would result in dissociation of the variable domains and abolishment of the affinity against VEGF. Hence the ratio reflects the percentage of interface disulfide bond formation in the sc-dsFv. This measurement was validated with the positive control (anti-VEGF scFv(fXa+)/M13pIII-pelB with -IEGR- (SEQ ID NO:599) in the linker peptide but without the interface disulfide bond) and the negative control (anti-VEGF scFv(fXa−)/M13pIII-pelB without both the fXa cutting site and the interface disulfide bond).

FIG. 6A compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the disulfide bond formation in the sc-dsFv displayed on phage surface for the signal sequence variants from the L4 library. Strong correlation between the two measurement is evident (R²=0.508, p-value=0.000158). As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule.

Another folding quality of the sc-dsFv was determined by the ratio of the normalized sc-dsFv-VEGF binding ELISA signal over the normalized quantity of the secreted sc-dsFv determined by electrophoresis and Western blot analysis. FIG. 6B compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the folding qualities derived from electrophoresis and ELISA measurements for the signal sequence variants from the L4 library. The positive correlation (R²=0.296, p-value=0.062) shown in FIG. 6B indicated that the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction. The plot also indicated that the selected variants resulted in secreted sc-dsFv with up to more than 10-fold VEGF-binding signals per unit quantity of secreted sc-dsFv compared with the positive control scFv(fXa+)/M13pIII-pelB, indicating that the secreted sc-dsFv from these signal sequence variants folded into antibody-like structure substantially more effectively that the scFv construct. This is most likely due to the stabilizing interface disulfide bond that is formed in the sc-dsFv but is absent in the scFv construct.

Example 7 Correlation Between the Stability of sc-dsFv and the Extent of the Interface Disulfide Bond Formation in the sc-dsFv

The effect of interface disulfide bond in stabilizing the sc-dsFv structure was demonstrated in FIG. 7. Secreted sc-dsFv from representative variants selected from each of the three libraries were expressed and incubated at 37° C. for 12 days and the affinities of the sc-dsFv's against VEGF were measured along the course of incubation. FIG. 7A shows the VEGF-binding affinity plotted against the time course of incubation for each of the selected variants. The VEGF affinity for the control anti-VEGF scFv dropped rapidly in the first few days of incubation, while a few variants from L4 library resulted in stable secreted sc-dsFv that were even gaining affinities against VEGF compared with freshly prepared secreted protein, presumably due to the increasingly stabilized sc-dsFv with the formation of the interface disulfide bond. The correlation between the two measurements shown in FIG. 7B is strong (R²=0.867 p-value=0.023), indicating that the interface disulfide bond could be one of the most important factors in stabilizing the secreted sc-dsFv in the culture medium.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims. 

What is claimed is:
 1. An isolated nucleic acid, comprising: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
 2. The nucleic acid of claim 1, further comprising a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence.
 3. The nucleic acid of claim 1, wherein the nucleic acid is an expression vector for expression a fusion protein containing the signal peptide and the single chain antibody.
 4. The nucleic acid of claim 1, wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond.
 5. The nucleic acid library of claim 4, wherein the first variable region is a heavy chain variable region (V_(H)) or a light chain variable region (V_(L)).
 6. The nucleic acid library of claim 4, wherein the second variable region is a heavy chain variable region (V_(H)) or a light chain variable region (V_(L)).
 7. The nucleic acid of claim 3, wherein the expression vector is a phagemid.
 8. A host cell containing the nucleic acid of claim
 4. 9. A phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface, wherein the phage is prepared by the method comprising the steps of: providing a host cell of claim 8, and culturing the host cell in a medium under conditions allowing expression of the phage.
 10. A method for producing a disulfide-stabilized single chain antibody, comprising providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody, wherein the expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3′ downstream to the first nucleotide, and wherein the signal peptide has the amino acid sequence of VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.
 11. The method of claim 10, wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond.
 12. The method of claim 11, wherein the first variable region is a heavy chain variable region (V_(H)) or a light chain variable region (V_(L)).
 13. The method of claim 11, wherein the second variable region is a heavy chain variable region (V_(H)) or a light chain variable region (V_(L)).
 14. The method of claim 10, further comprising, after the culturing step, collecting the medium for isolating the disulfide-stabilized single chain antibody.
 15. The method of claim 10, wherein the expression construct is a phagemid that further includes a third nucleotide encoding a phage envelope protein, the third nucleotide sequence being located 3′ downstream to the second nucleotide sequence.
 16. The method of claim 15, further comprising, after the culturing step, collecting the medium for isolating phage particles that display the disulfide-stabilized single chain antibody. 